IL102079A - Use of CANP-inhibitors in pharmaceutical preparations - Google Patents

Abstract

The invention relates to the use of inhibitors of neutral Ca<2+> dependent proteinases (CANPs) and their pharmaceutically acceptable addition salts or their active subunits in the field of tumour therapy, especially cancer therapy, viral diseases therapy, AIDS therapy and contraception. The inhibitors can especially be used against protease bound glycosaminoglycan-substrate dependent tumours, viral diseases and contraception by selective malignant cell killing, viruses and spermatozoa killing and inhibiting of nuclear vlimmata in a host-parasite cell system involving common protease bound substrate. The inhibitors are heatstable, tetrameric proteins at neutral pH, approximatively 240 KDa, destroyed with trypsin. The pharmaceutical compositions may be prepared in a manner known per se, with carriers or additives commonly used in the pharmaceutical industry.

Description

Π π ΊΊ >ν¾ηηι CANP >n ^Ql ¾ Ί» ¾J -INHIBITORS IN PHARMACEUTICAL PREPARATIONS USE OF CANP-INHIBITOR IN PHARMACEUTICAL PREPARATIONS FIELD OF THE INVENTION The present invention relates to the pharmaceutical use of specific inhibitors of Calcium Activated Neutral Proteases (CANPs) or its active subunits. More specifically, the inhibitors or its active subunits may be especially used for the treatment of tumours, especially cancer, viral diseases, AIDS, and as anticonceptiva. The invention further relates to the manufacture of pharmaceutical preparations for the treatment of the above mentioned diseases and for the use as anticonceptiva.

BACKGROUND OF THE INVENTION One of the most lethal properties of malignant cells is their ability to infiltrate normal tissues and to metastasize to distant areas. The normal connective tissues consist of cells embedded in an extracellular matrix containing glycoproteins, collagen, elastin and proteoglycans . There have been made suggestions that tumour associated histolytic enzymes may aid in the invasive process by removal of the matrix protein (Hart, I. et al., 1980, JNCI 64:891). Several studies have concentrated on this aspect of tumour cell biology, and increased protease production has been observed with many transformed cells (Jones P.A. and Declerk Y.A., 1980, Cancer res. 40:3222). m- and μ -calcium-activated neutral proteases (CANPs), also known as calpain I and II, are typical intracellular cysteine proteinases of higher animals. They have been presumed to participate in various cellular functions mediated by Ca^+, but their precise function are not yet clear. CANPs hydrolyse proteins of limited classes in vitro, including epidermal growth factor receptor, platelet derived growth factor receptor and protein kinase C. They inhibited (Mehdi S. et al., 1988, Biochem. and Biophys. Res. Comm. 157(3) 1117-1123) or only partially inhibited by leupeptin (Tsuji S. and Imahori , 1981, J. Biochem. 1990, 233-240). In addition, the substrate in malignancies upon which the inhibitors of CANPs act were unknown until now. Moreover, as will be reported later, leupeptin and E64 used in tests according to the present invention did not inhibit malignant cell growth.

It has been published that malignant cells in culture from human invasive urothelial carcinoma form tumour nodules and glycosaminogl can membranous sacs (GSG) with membrane extensions intracellularly as well as extracellularly (Logothetou-Rella, H. et al., 1988a, Europ. Urol. 14(i), 61-64 and 65-71). The same observations were made in human trophoblast cell cultures (Logothetou-Rella, H. et al., 1989, Histol. Histopath. 4:367-374), while they were not found in human normal urothelial cells in culture (Logothetou-Rella, H. et al., 1988, Europ. Urol. 15, 259-263). The participation of GSG has also been reported in capillary formation which is enhanced in tumours in vivo (Lo-- gothetou-Rella, H. et al., 1990, Histol. Histopath. 5:55-64).

The characteristic extracellular matrix (GSG) of malignant and embryonic cells is PAS and PAS-diastase positive, identified by Papanicolaou stain by its light green colour (EA colour) and smooth, to fibrillar translucent texture. GSG in malignant cells is distributed and accumulated in intracellular and extracellular membranous sacs. The membranous GSG sacs give rise to membrane extensions which form channels through which the green GSG is passed from the inside to the outside of the cell, enhance tumour nodule formation and invade other cells in vitro.

OBJECT OF THE INVENTION Surprisingly a new mechanism of cell to cell invasion and substrate (GSG bound CANP) formation, common e.g. in formation of tumours, viral diseases, AIDS and fertilization were found.

Moreover it was found that administering specific inhibitors of CANPs or an active subunit of it to provide an effective concentration of said inhibitors in human or animal body would inhibit the aforementioned processes.

DETAILED DESCRIPTION OF THE INVENTION It was found that the intracellular and extracellular matrix (sac-GSG bound CANPs) produced by the interaction of malignant with normal cells in vitro and in vivo can be used as substrate for the specific inhibitors of CANPs or its active subunit.

The intracellular GSG bound CANPs-sacs communicate with the extracellular environment with membrane extensions and form large extracellular channels (full of substrate) or diffused matrix allowing the large molecule (approximately MW 240,000) of the inhibitors of CANPs to also enter the cells and inactivate GSG bound CANPs. The sac-GSG bound CANPs and extracellular matrix e.g. in tumours are produced by a new mechanism of cell to cell invasion related to that of viral cell infections and fertilization. The inhibitors of CANPs selectively kill malignant cells only by inacti-vation of the intracellular and extracellular GSG bound CANPs, a special matrix upon which viability and propagation of only malignant cells depends on.

The viral infection of cells and oocyte penetration by spermatozoa are biological phenomena which also involve cell to cell invasion, i.e. cell invasion by virus and oocyte invasion by spermatozoa, produce the same extracellular matrix (substrate) as malignant cells and require the presence of CANPs. Based on this new mechanism of cell to cell invasion, the inhibitors of CANPs or its active subunits also exhibit antiviral and contraceptive action.

A possible mechanism of the action of an inventive inhibitor on malignant cells might be e.g. the dissociation of the inhibitor tetramer, upon contact with extracellular GSG (substrate) bound CANPs, into subunits (e.g. monomers, the MW of which vary considerably and depend on the substrate used) and formation of inactive inhibitor-proteinase complex (blue hematoxyl nophic granules).

Then the following events may be taking place. The inhibitor subunit produced extracellularly may diffuse through malignant cell membrane and inactivate the endogenous CANPs or the intracellular activated CANPs diffuse extracellularly towards a lower concentration gradient (after the inactivation of the extracellular activated CANPs) and get inactivated by the extracellular inhibitor monomer. Also both events might be taking place. Empty cytoplasmic vacuoles observed in inhibitor treated malignant cells support the diffusion of activated CANPs and its inactivation extracellularly.

Moreover the extracellular GSG-CANPs channels are large enough for passage of the inhibitor all the way into the GSG-CANPs sacs.

The effect of the inhibitors of CANPs on spermatozoa suggests that spermatozoa are associated with the CANPs enzyme, upon which their motility, viability and penetration ability depends. The increased sperm motility observed with high Ca2+ concentration (Fakih et al., 1986, Fertil. Steril. 46(s), 938-944) could be achieved via the activation of sperm endogenous CANPs. The high Ca2+ release by the egg cortex upon polyspermy prevention ( Steinhardt et al., 1977, Develop. Biol. 58, 185-196) might involve the participation of the oocyte's possible inhibitors of CA Ps.

The cytologic effect of the inhibitors of CANPs on semen mucin strongly documents that the active enzyme is bound to glycosaminoglycans, an apparently common substrate for both the protease and inhibitor action. Again the mechanism of action of the inhibitors on spermatozoa might be the dissociation of a certain inhibitor upon contact with mucin (substrate) bound CANPs and formation of inactive inhibitor -proteinase complex (blue hematoxylimorphic granules). The inhibitor subunit produced extracellularly after dissociation, is probably diffused through spermatozoa membrane and inactivates the endogenous CANPs or CANPs moves ex-tracellullarly towards a lower concentration gradient after inactivation of extracellular CANPs. The inhibitors of CANPs being non-toxic to normal cells and toxic to spermatozoa seems a promising male contraceptive agent.

This observation enables a new approach to many pharmaceutical problem, but especially enables the manufacture of new pharmaceutical preparations containing suitable inhibitors of CANPs for the treatment of tumour, especially cancer, all kind of viral deseases, AIDS and fertilization and methods for the treatment of the aforementioned pharmaceutical diseases and needs.

The inhibitor of CANPs used in the invention is preferably a tetrameric protein of MW of approximatively 240,000, based on its elution from Sephadex G-200, heatstable at neutral pH, destroyed on digestion with trypsin, and dissociated into its subunits of a MW of approximatively 60,000 by 0.1-1 mM Ca2+, based on SDS-polyacrylamid gel electrophoresis (as described by Melloni et al. in Arch, of Biochem. and Biophys. Vol. 232, No. 2, 513-19, 1984). All pharmaceutical acceptable salts, derivatives, analogues or active subunits of different MWs (which depend on the substrates used) thereof can also be used as specific inhibitors of CANPs.

It is believed that the MWs of the active subunits of the specific inhibitor depend on the substrates (e.g. Casein, denatured globin etc.) used. Therefore, the MW of the active part of the inhibitor may be higher, e.g. approxi-matively 150,000, or lower, e.g. approximatively 15,000, as indicated above.

The inhibitors may be endogenous native inhibitors isolated from a biological source, like erythrocytes, brain, cardia muscle, lung, spleen, liver, skeletal muscle, kidney, testis or the like, and optionally purified, but especially from rabbit skeletal or liver.

A preferred inhibitor isolated from rabbit skeletal is manufactured and sold by Sigma Chemical Company, St. Louis, USA, under the product number P-0787.

The inhibitors or active fragments thereof, like subunits of MG 60,000, may also be produced synthetically, especially by bio- or gentechnological methods, e.g. by expression in Escherichia coli.

In the present invention the pharmaceutical preparation may be in the form of a solution, powder, injection, tablet, capsule, pellets, in a fast or sustained release form, each containing a suitable amount of a specific native or synthetic and eventually purified inhibitor or its pharmaceutically acceptable addition salts, active subunits, fragments, derivatives, or related compounds together with well-known suitable excipients.

The inhibitors may preferably be administered to humans and warmblooded animals intramuscularly, subcutaneously, intraperitoneally or intravenously in an amount which depends on the kind and severity of the disease, the inhbitory effect of the inhibitor, the route of administration, the species to be treated, the weight and the general condition of the patient, and has in most cases finally to be decided by the responsible physician. In general the dose is between about 1 mg/kg per day and 25 mg/kg per day. However, if need be also higher doses, e.g. up to 100 mg/kg per day, may be administered.

The surprising effect of the inhibitors of CANPs have been confirmed and verified by the following tests, whereby all tests have been fulfilled also with Aprotinin (Sigma A-4 529), Trypsin-Chymotrypsin inhibitor (Sigma T-9777), Leupeptin (Sigma L-2884) and E64 (Sigma E-3132) dissolved in RPMI-1 640 with 25 mM hepes as control protease inhibitors. All inhibitor solutions were filtered through 0.22 μ Sar-torius filters, dispensed in aliquots and frozen at -20° C. Fresh or thawed inhibitor solutions were used.

However, the surprising effect was only performed by the inhibitors of CANPs according to the invention. In the following tests the brownish tan powder of the inhibitor of CANPs (Sigma Chemical Company, St. Louis, USA, product number P-0787), 50 U/645 mg solid from rabbit skeletal muscle, was dissolved in 5 ml plain RPMI-1640 with 25 mM hepes (Seromed), resulting to a clear tan solution (10 U/ ml), whereby one unit (U) is that quantity of inhibitor which will reduce the activity of 1 unit of CANPs (Sigma Chemical Company, product number P 4533) by 50 % at pH 7.5 at 30°C (reaction volume = 1.8 ml, 1 cm light path). Of course, the scope of the invention shall not be reduced to the use of the concrete inhibitor used in the following examples .

EXAMPLE 1 The use of the native Inhibitor of CA P for inhibiting growth and viability of malignant cells in Vitro Cell culture establishment Stationary cell cultures were established from human solid tumour tissue specimens by enzymatic digestion. Malignant lung cell lines from metastatic lung carcinoma, M-cells, P-cells and B-cells have recently been characterized by the applicant. Malignant urothelial cell cultures were established from tissue specimen from patients with invasive transitional cell carcinoma. The five established urothelial malignant cell lines were designated as Pa-cells, R-cells, S-cells, Br-cells and IG-cells. Only the patient where Pa-cells derived, had received bladder intravesical infusions of anticancer drugs. Melanoma cell culture (Ha-cells) was originated from a male patient who suffered from primary rectal melanoma, metastasized at the lymph nodes of the right arm where tissue specimen was obtained.

Malignant bone marrow cells were originated from bone marrow aspirates from five (5) patients with chronic myeloid leukemia. Walker tumour rat cells were isolated from transplanted tumour tissue into Wistar irats. Normal human liver cells (L-cells) were isolated from liver tissue specimen from a male patient who underwent surgery for the removal of his gall bladder.

Normal fallopian tube cells (F-cells) were isolated from tissue specimen from a female patient who underwent total hysterectomy. Normal bladder cells (N-cells) have been characterized previously (Logothetou-Rella, H. et al., 1988, Europ. Urol. 15, 259-263). White blood cells from five healthy persons were also used as control cells .

Mice embryos harvested at the 2-cell stage were cultivated in complete Earle's balanced salt solution (EBSS supplemented with 10% foetal bovine serum and antibiotics ) to the stage of hatched blastocysts. When the embryonic cells were all out, the culture was used for cytology. Amniotic ebryonic cells from five (5) pregnant women cultivated for prenatal diagnosis were also used in this study. All cell cultures were grown in complete medium, RPMI-1640 (Seromed) supplemented with 10% foetal bovine serum (Seromed), glutamine and antibiotics (Seromed), incubated at 37 °C in a C02~humidified incubator. Stock cells are stored frozen in liquid nitrogen.

Cytogenetic analysis Chromosomal analysis of M-cells, P-cells and B-cells have recently been reported (Logothetou-Rella, H. et al.,1991, J.Exper. Clin. Cancer Res., submitted for publication). Urothelial malignant, Pa-cells consisted of malignant cell clones only, with polyploidies up to 147 chromosomes and complex structural abnormalities. S-cells consisted of malignant cell clone with regular tetraploidies, up to 20% of the cell population and 80% normal cell clone. Br-cells consisted of normal and malignant cell clones but detailed chromosomal analysis was unsuccessful. Melanoma Ha-cells revealed only double minutes. Liver L-cells, fallopian tube F-cells, and amniotic embryonic cells were cytogenetically normal .

Two techniques were used to determine the inhibitor's cytotoxicity on tumor and normal cells. a) Cytologic changes of cell cultures in continuous presence of the inhibitors Seven kinds of complete medium RPMI-1640 were prepared. One was supplemented with 1 U/ml of the inhibitor of CANPs; the second with 2 mg/ml trypsin-chymotrypsin inhibitor; the third with 1 mg/ml aprotinin; the fourth with 1mg/ml leupeptin; the fifth with 1mg/ml E64; the sixth with all five inhibitors at the same concentration and the seventh complete RPMI-1640 as a control medium.

Ten glass petri-dishes (5 cm diameter) were seeded each with 1x10*> M-cells and another ten dishes, each with 1x10> P-cells. Duplicate cell cultures received each kind of complete medium containing the inhibitors and control cultures containing only complete medium. The cell cultures were incubated at 37 °C in a humidified CC>2-inhibitor for 120 hours. The culture medium was changed with a fresh one of the same kind in each case, 24 and 72 hours after culture initiation. Half of the cell cultures were fixed in 50% ethanol 72 hours and the other half 120 hours after culture initiation. All cell cultures were stained with the Papanicolaou method.

Post confluent stationary cell cultures of malignant M-cells, P-cells and normal L-cells (20 days of continuous cultivation) that had produced abundant extracellular matrix, received fresh complete medium RPMI-1640 supplemented with 1 U/ml of the inhibitor of CANPs and incubated at 37 °C for 3 days, then fixed in 50% ethanol and stained with the Papanicolaou method.

Trypsin-chymotrypsin inhibitor, aprotinin, leupeptin and E64, did not affect the growth and cytology of M- and P-cells as compared to control cell cultures.

The inhibitor of CANPs caused great exfoliation of cells and extracellular matrix (ECM) in the culture medium, after 72 hours of continuous presence in cultures. All exfoliated cells were dead (according to trypan blue stain) consisting of hyperchromatic, pyknotic nuclei, little cytoplasm and nuclei with tails. On the culture dish surface, a few, countable per field, attached fibroblast-like cells remained alive, cytologically normal. All other cell culture dishes (except cases No. 1 an No. 6 which contained the inhibitor) and the control ones were full of cells and nuclear vlimma (="NV", "vlimma" = bullet; state of a parasitic cell after numerous dividing operations using a host cell, where it reaches the size of a nucleolus with a nuclear head and an attached tail resembling a small immotile spermatozoo. The production of nuclear heads, the shooting and implantation in other cells occurs mainly in culture areas, where extracellular glycosaminoglycans bound CANP and cell membranes are located. In normal cell cultures NV are not found, whereas it was observed e.g. in human solid and hematologic tumours free or during production still attached to the mother cell. NVs are end cell products of incomplete, unequal, asymmetrical division of malignant cells. Upon their production they eventually detach from their mother cell and seek a host cell at random. When NVs are implanted and incorporated in the nucleus of a normal host cell, it can be considered as a process similar to fertilization or viral infection. As a result, the host cell's genotype and phenotype is altered and behaves like a transformed cell. After many - divisions, the host cell looses its cytoplasm and cannot divide itself anymore; it needs support by another host cell or extracellular matrix, thus forced to become a parasite and produce NVs.) uncountable per field, without cell exfoliation, with macroscopically apparent green, fibrillar, translucent ECM and GSG sacs. The observations were persistent after 120 hours of continuous presence of the inhibitor of CANPs in cell cultures, except that the survived fibroblast-like cells had grown up in the presence of the inhibitor of CANPs.

Post-confluent M- and P-cell cultures exhibited cells with vacuolated cytoplasm as a strain, and degenerated nuclei of different sizes with and without tails. The rounded up, detached, dead cells were holding to each other on the culture dish surface by a network of hematoxylinophilic (blue) membranes visible microscopically. The EC and GSG sacs had disappeared. Instead large masses of hematoxylinophilic granules were present visible microscopically. b) Liquid medium short-term culture method (Chang S.Y. et al, 1989, Eur. Urol. 16, 51-56).

The cells were detached with trypsin-EDTA (Seromed) resuspended in complete RPMI-1640 and cell counts were made using a hemocytometer . Viable counts were assessed using the 0.4% trypan blue exclusion method. The cells were then washed once with complete RPMI-1640, centrifuged at 200 g for 8 min, resuspended in complete RPMI-1640 at 30,000 -200,000 cells per 0.5 ml medium and inoculated in polypropylene tubes as shown as follows: Test nCANP Volume (ml) tube inhicase bitor nCANP Cell Complete No. (U/ml) inhibitor suspension F.-B.S. * RPMI-1640 1 1 0.1 0.5 0.4 2 2 0.2 0.5 - 0.3 3 3 0.3 0.5 - 0.2 4 4 0.4 0.5 0.05 0.05 5 5 0.5 0.45 0.05 - 6 6 0.6 0.35 0.05 - 7 0 0.0 0.5 _ 0.5 *F.B.S. = Foetal bovine serum Duplicate samples of cells were tested for each concentration of the inhibitor. All samples were incubated and shaken in a water bath at 37° C for one hour. Then the cells were washed twice with complete RPMI-1640 by centrifugation at 200 g for 8 min. Each rinsed cell pellet was resus-pended in 1 ml complete RPMI-1640, the cells were then rendered single by gentle pipetting and were then seeded in 24-well microplates (Costar Cambridge Mass.) for a 4-day period of short term culture at 37 °C under a humidified atmosphere of 5% C02« The cytotoxicity assessment was done using the dye exclusion method of 0.4% trypan blue. The degree of cytotoxicity was measured according to the following formula: Number of viable cells in the experimental group Cytotoxicity (%) = 1 - x 100 Number of viable cells in the control group The inhibitor of CANPs selectively killed all kinds of malignant cells tested (Table 1 ) while allowing normal cells within the same or separate culture to grow and propagate (Table 2). The optimum concentration of 4-5 U/ml inhibitor killed all malignant clones, while lower concentration killed lower percentage of malignant cells . Higher concentration did not alter the results . The inhibitor was not cytotoxic to normal cells including liver cells, fallopian cells and WBCs. Cytogenetic analysis of the survived cells (in mixed cell lines) after the inhibitor CANPs treatment showed normal karyotype. The inhibitor of CANPs was also cytotoxic to embryonic cells.

Table 1. Categories of cases with various cells tested.

Malignant Tissue origin Designa- and Malignant Normal tion normal clone clone clone Bladder transiPa-cells tional cell Br-cells + carcinoma S-cells + IG-cells R-cells + Lung carcinoma M-cells + P-cells + B-cells + Melanoma Ha-cells Chronic myeloid leukemia BM-cells Walker rat tumor W-cells Normal liver L-cells + Normal urothelium N-cells + White blood cells WBC + (5 specimens) Human amniotic embryonic cells (5 specimens) + Human fallopian cells (F-cells) + +: Indicates the cytogenetic state of each cell type.

Table 2. Sensitivity of cells to different concentrations of the inhibitor of CA Ps.

Inhibitor cytotoxicity (%) Tested cells Inhibitor-nCANP (U/ml) 1 4 5 M-cells 24 65 65 P-cells 45 82 B-cells 86 87 Pa-cells 24 99 100 S-cells 34 Br-cells 55 R-cells 83 IG-cells 100 Ha-cells 21 100 Walker tumor cells 100 Malignant bone marrow cells 88-100 N-cells 0 0 0 L-cells 0 0 0 WBCs 0 0 0 Embryonic cells 45 95 100 F-cells 0 0 0 The cytotoxicity of the inhibitor in each specimen was obtained from mean of duplicate samples.

EXAMPLE 2 Use of the inhibitor of CANPs on the viability of normal and malignant urothelial tissues Tumour (from 5 patients) and normal (from 5 persons) tissue pieces of human urothelium of 2 mm x 2 mm x 2mm size were rinsed in complete RPMI-1 640 , handled gently with fine forceps, immersed one piece (of each type of tissue) in complete RPMI-1 640 (control) and one piece in the inhibitor solution ( 1 0 U/ml) in polypropylene tubes and incubated at 37 °C for one hour in the humidified, 5% CO2 incubator. All tissue pieces were then rinsed carefully in complete medium and were immersed in polypropylene tubes ( 1 piece/tube) containing 2 ml complete RPMI-1 640 , and then incubated for 4 days at 37 °C. The tissue pieces were fixed in formaldehyde, embedded in paraffin and tissue sections were stained with eosin-hematoxylin. The exfoliated cells in the tubes with the malignant tissue pieces were allowed to settle in a conical polypropylene tube for 1 0 min, then smeared on glass slides fixed with cytospray and stained with Papanicolaou. The inhibitor of CA Ps caused massive cell exfoliation of the malignant tissues. Histologic examination of the inhibitor treated malignant tissues exhibited bionecrotic to necrotic areas and large tissue areas consisting of eosinophilic extracellular matrix denuded of cells. The exfoliated cells were dead, with degenerated nuclei, and spermatozoa-like morphology, separated from each other and lacking the green ECM. The very few malignant tissue exfoliated cells, in the absence of the inhibitor of CANPs, showed compact cell masses in green ECM with indiscrete cell boundaries.

Normal urothelial tissues were kept intact after treatment with the inhibitor.

EXAMPLE 3 The use of the inhibitor of CANPs against human tumour nodule in vivo A female patient with breast metastatic carcinoma, with extensive hepatic bone and subcutaneous metastasis, after repeated chemotherapy and radiation treatment without success, and also in a general bad health condition, approved the trial of the drug in her subcutaneous nodules. Nodules were located all over the chest and some in the abdomen. One hard nodule the size of a pea was injected in its center with 0.1 ml (1 u/0.1 ml) inhibitor of CANPs dissolved in RPMI-1640 with 25 mM hepes. Twenty four hours later the treated nodule and a nearby untreated control nodule were removed, fixed in formalin and embedded in paraffin for microscopical examination.

The patient did not show any allergic reaction 1 , 4 and 24 hours after the injection. The nodule feeling was softer and slightly smaller 4 hours after the injection. Twenty four hours later the nodule was soft and reduced to half its originial size. The treated neoplasm was histologically characterized by degenerated small cellular aggregation and many degenerated single cells. Most of the cells had irregular pyknotic, karyolytic or degenerated vacuolated nuclei. Some- cells showed vacuolated cytoplasm. In the outer peripheral area of the tumour there remained a few neoplastic cells with regular nuclei, fine chromatin and slender single nucleoli . The main area of tumour cell degeneration, caused by the inhibitor of CANPs, was measured approximately to a total of 3.4 mm x 2.5 mm out of the 5.2 mm x 2.5 mm main tumour section area. Neighboring sweat glands and the overlying epidermis were kept intact. There was no inflammatory reaction not even around the fissural hemorrhagic area caused by the injection. Histologic examination of the tumour nodule without treatment showed that the neoplasm was characterized by viable large columns or single strands of neoplastic cells with relatively uniform ovoid or roundish nuclei with finely stippled chromatin and slender nucleoli. Histologic picture was compatible with metastatic breast carcinoma.

EXAMPLE 4 The use of the inhibitor of CANPs against rat tumours in vivo Two Walker tumours were excised 2 weeks following the subcutaneous implantation of tumour tissue in Wistar male rats. Tumour cell suspension for injection was prepared as described previously (Fisher E.R. and Fisher B., 1959, 12, 926-928). A group of Wistar male rats, weighing 100 g each, were injected with 10x10^ Walker tumour cells subcuta-neously in the left foot pad. The rats were then divided into four groups , two control and two treated . Treatment was initiated when tumours had reached a measurable size of 50-100 cu.mm. The first group of rats was injected intra-peritoneally , each rat with 50 U/2.5 ml (645 mg/2.5 ml) inhibitor of CANPs, once a day, for a period of 2 days (0.5 U/kg or 6.45 mg/kg rat body weight).

The second group of rats was treated intraperitoneally, twice daily for 5 days with the dose of 0.25 U/kg (3.23 mg/kg) rat body weight. Control rats were injected each with 2.5 ml Medium RPMI-1640 with 25 mM hepes. All rats were sacrificed 4 days after the last treatment for the injected legs, of the control groups, were all covered with tumours including up to the shoulder blade and accurate control tumour measurements were impossible. The tumour- legs, lymph nodes and liver from all rats were excised, fixed in formalin and embedded in paraffin for histologic studies. Tumour volumes were measured every day after the first dose, with calipers. The inhibitor of CANPs caused 50% tumour regression in the first group of treated rats and 90% in the second group. All groups (treated and control) started at time 0 without any significant difference in tumour volume .

The rats under treatment were healthy and did not show any allergic reaction or side effects to the high dose of the inhibitor originating from rabbit skeletal muscle. Histologic examination of livers of the treated rats did not show any cytotoxic effects caused by the inhibitor, as central venules were observed without necrosis or cellular damage .

Among the first treated group one rat developed metastatic abdominal focus and another one metastatic hepatic focus. The feeling of the abdominal focus disappeared 24 hours after the first dose. Histological examination showed necrosis of large carcinomatous nodule with formation of abscess, necrosis of the overlying epidermis and ulceration. The liver metastatic focus was necrotized, exhibiting necrotic material with nuclear debris in the center and remnants of carcinomatous tissue with mitoses in its periphery. The foodpad tumours of treated rats showed necrotic areas of variable size with formation of microabscesses. These results become more important if taken into account the aggressiveness of Walker tumor cells, (rats usually die 20 days after tranplantation) .

CONCLUSION DEDUCED FROM EXAMPLE 1-4 The pharmaceutical composition, of the present invention killed all malignant cells of different chromosomal abnormalities, tissue and species origin without affecting normal cells' genotype.

The best in vitro dosage being the one containing 4-5 units of the inhibitor of CANPs per milliliter of solution.

This pharmaceutical composition was not cytotoxic to normal cells including liver and WBCs. It exhibited a broad spectrum of action on different types of human tumours. It was cytotoxic to human solid and hematologic tumour cells and even more to chemoresistant tumour cells (lung P-cells and bladder Pa-cells) of different tumour origin (embryonic cells were very sensitive to the inhibitor because of their resemblance to malignant cells).

The pharmaceutical composition of the present invention tested on rat tumours caused 50-90% regression of the main tumours, inhibited metastasis and caused necrosis of metastatic foci. It was non-immunogenic, non-toxic suitably used in a daily dose of 0.5 U/Kg, (6.5 mg/Kg) of body weight in single or divided doses. The administration is suitably continued until complete tumour regression.

EXAMPLE 5 Contraceptive action of the inhibitor of CA Ps Fertile motile sperm (after swim up test) from 10 donors, was dispensed in 4 plastic (5 ml, Falcon) test tubes each. Two test tubes received 0.4 ml sperm suspension (600,000 spermatozoa) and 0.6 ml complete EBSS. The other two test tubes each received 0.4 ml sperm suspension, 0.4 ml (10 U/ml) inhibitor of CANPs and 0.1 ml foetal bovine serum. All tubes were incubated at 37 °C for 1 hour. The spermatozoa were then washed twice with complete EBSS and cen- trifuged. The sperm pellets were resuspended each in 2 ml complete EBSS and incubated at 37 °C for 18 hours, at which time spermatozoa were counted by the Eosin Y viability exclusion stain, smeared on glass slides, fixed in 50% ethanol and stained with Papanicolaou.

The degree of cytotoxicity caused by the inhibitor of CANPs was measured according to the following formula: Number of viable inhibitor treated spermatozoa Cytotoxicity (%) = 1 - X 100 Number of viable spermatozoa of control samples Inhibitor treated and non treated sperm was inoculated in postconfluent granulosa cell cultures, incubated at 37° C for 18 hours, fixed in 50% ethanol and stained with Papanicolaou. Motility counts of the inhibitor treated fertile sperm showed only 3% motile sperm, 2 hours post treatment and no motile sperm 18 hours post treatment. Eighty percent of the immotile sperm were dead (stained with eosin Y) . Inhibitor treated sperm failed to penetrate granulosa cells during co-cultivation.

Cytologic examination revealed that 80% of the inhibitor treated spermatozoa, 18 hours post treatment, had coiled tail ends and clear acrosome caps. Moreover, the green fibrillar mucin present in the control samples was changed to dispersed loose masses of large blue granules in the inhibitor treated spermatozoa.

EXAMPLE 6 Antiviral action of the inhibitor of CANPs in vitro 1 ) On Eps ein-Barr Virus (EBV) Viral infection of cells which involves host-parasite interaction. Viruses are also vehicles of biological active DNA or RNA into host cells in order to survive and propagate, causing viral diseases to animals and humans.

In the following experiments the CANPs inhibitor was tested for its antiviral action in two cell lines . One cell line was cultured Burkitt tumour lymphoblasts (strain Raji) infected with Epstein-Barr virus (EBV) in vitro (Kottaridis et al, J. Natl. Cancer Inst. 1977 , 59 ( 1 ) , 89-91 ) and the other P3HR-1 Burkitt lymphoma, EBV producing cells (Hinuma Y. et al, 1967 , J. Virol. 1 , 1 045-1 051 ) . IgG antibodies were demonstrated by immunofluorescence (Gull labs) on EBV-infected Raji cell smears with 1 0% and on P3HR-1 with 20-25% of the cells showing fluorescence. For negative controls IgG-EBV negative antibodies were used.

For chemosensitivity testing 200,000 cells/tube were used. The results for EBV-infected Raji cells showed 22% cytotoxicity at 2 U/ml CANPs inhibitor, 97% at 4 U/ml and 100% at 5 U/ml. In the case of P3HR-1 , EBV producing cells, there was 95% cytotoxicity at 2 U/ml CANPs inhibitor, 100% at 4 u/ml and 5 U/ml. Both cell lines were found free of detectable immunofluorescent IgG after treatment of cells with higher than 4 U/ml CANPs inhibitor.

Although both cell lines are sensitive to the CANPs inhibitor, because they are malignant, the disappearance of immunofluorescent IgG after treatment documents the antiviral action of the CANPs inhibitor. 2) On Human Immunodeficiency Virus Type 1 (HIV-1 , AIDS Virus) The cell line MOLT-4 (ATCC CRL 1582, from acute lymphoblastic leukemia) infected with HIV-1 (Koyanagi Y.S. et al., 1987, Science 236, 819-822 and Cann A.J. et al., 1990, J. Virol. 64 (10) 4735-4 742) was used. For chemosensitivity testing 200,000 cells/tube were treated with the inhibitor of CANPs at 4 U/ml and 10 U/ml. The results showed 97% cytotoxicity at 4 U/ml and 100% at 10 U/ml inhibitor of CANPs.

Cell smears, fixed in cold acetone, were used for immunofluorescent tests, using positive human serum containing antibodiesto HIV-1 (1:100) and antihuman IgG fluorescent conjugate (1:200 Dako corp.). For negative controls human serum negative to HIV-1 antibodies was used. Immunofluorescent HIV-1 -antigen was detected in 60% of untreated MOLT-4 HIV-1 infected cell cultures. The treated MOLT-4 HIV-1 infected cells were free of detectable immunofluorescent HIV-1 -antigen at concentrations of 4 U and 10 U/ml inhibitor of CANPs.

It is concluded that HIV-1 infected MOLT-4 cells are highly sensitive to the inhibitor of CANPs for they are malignant cells but the disappearance of immunofluorescent HIV-1 -antigen documents the anti-AIDS action of the inhibitor of CANPs.

The use of CANP inhibitors against viral diseases .

Epstein-Barr virus (EBV) infected Raji cells, EBV producing P3HR-1 cells (Kottaridis et al, 1977, J. Natl. Cancer Inst.59( 1 ), S9-91 ) and HIV-1 infected molt-4 cells (koyanagi et al, 1987, Science 236, S 19-S22) grown in RPMI- 1640 supplemented with 10% fetal borine serum and antibiotics were cept in humidified CO2 incubator at 37°C. Each cell type ( 100.000-200.000 cells) was treated according to the liguid medium short-term culture method ( Chang et al 19S9, Eur Urol 1 6, 5 1 -56) with 4-5 u/ml native CANP inhibitor ; 0.4-0.8 mg/ml Calpain inhibitor I ; and 0.8- 1 .6 mg/ml Calpain inhibitor II. At 4 days post treatment cell survival and immunofluorecent VCA and HI V-I antigens were detected.

The results showed that the native CANP inhibitor and calpain inhibitor I caused 100% cytotoxicity to all cell lines treated and complete disappearance of VCA and HIV-I antigens at 4-5 mg/ml and 0.4-0.8 mg/ml respectively.

Calpain inhibitor II caused 100% cytotoxicity to all treated cell lines at 0.8- 1 .6 mg/ml but presence of weak positive imnumofluorescent VCA and HIV-I antigens.

The cytotoxicity and disappearance of immunofiuorcscent VCA and HIV-I antigen after treatment, documents the antiviral action of CANPS inhibitors ; the native CANP inhibitor and Calpain inhibitor I were more effective than calpain inhibitor II ( Logothetou-Rella, 1995 Histol Histopathol. 10, 27 1 -282).

Example S The use of CANP inhibitors as spermicidals and contraceptives.

Fertile motile ( 600.000) spermatozoa (after swim up test) from 10 donors were treated according to liquid medium short-term culture method ( Chang et al 1989, Eur Urol 16, 51 -56) with or without 10 u/ml native CANP inhibitor for 1 hour. Washed spermatozoa were incubated at 37°C in humidified CO incubator , in Earle's Balanced Salt Solution (EBSS) supplemented with 1 0% fetal bovine serum, pyruvate and antibiotics for I S hours. Then spermatozoa viability was estimated by the Eosin Y viability exclusion slain.

The results showed that the native CANP inhibitor caused S0% spcrmatozootoxicity documenting the spermicidial and hence contraceptive action.

The use of CANP inhibitors against spermatogenesis (meiosis).

Four groups, each of five male Wistar rats, were used for testing CANP inhibitors. The first group received 0.25u CANP inhibitor (Sigma, P-07S7) per gr body weight i.p. daily for six days. The second group received 0.27 mg Calpain inhibitor I (in liposomes), the third 0.27 mg Calpain inhibitor I I (in liposomes) per gr body weight i.p. daily for six says and the fourth control group, injections of salinc-fillcd liposomes. The following day after the last treatment, all rats were sacrificed and all organs including the testis were removed and examined histologically.

Histological examination of the testis of the rats treated with CANP inhibitors showed spermatogenctic arrest. The seminiferous tubules were devoid of spermatozoa and spermatids with degeneration of secondary spermatocytes and presence of degenerated spermatozoa in the lumen. The placebo rats showed testis with intact spermatozoa, spermatids and spermatocytes in all seminiferous tubules. All other organs of treated and placebo rats showed no histological toxic effects.

The results show the CANP inhibitors are cytotoxic to spermatocytes dividing by meiosis and to spermatids and spermatozoa ( identical to NVs of malignant cells), cellular products of meiosis . Hence, CANP inhibitors arc antimciotic agents.

Example 10 The effect of CANP inhibitor on survival of mice bearing L 12 1 0 leukemia /// vilro continuous treatment of murine L 1210 leukemia cells with 0.25 U/ml CANP inhibitor induced a significant and iarge cytotoxic effect. Therefore, 1 x 10^ L I 210 cells were implanted i. p. into female DBA/2 mice. Treatment with 2 U CANP inhibitor/mouse/day began one da subsequent to tumor cell implantation. Mice were then treated daily by an intraperitoneal injection of CANP inhibitor until the time of death. The median life span (MLS) of placebo-treated mice was calculated to be 1 1 .5S,± 0.34 days, while that of CANP inhibitor-treated animals was 1 3.58 ± 0.5 1 days (p=0.0036). This slight, although significant, increase in MLS is consistent with the /// vilro sensitivity of L 12 10 cells to the inhibitor of CANP.

Example 1 1 The effect of other CANP inhibitors on malignant cell survival Human cells of the established line NHIK 3025, originating from a cervical carcinoma /'// silii (Nordbye, . and Oftcbro, ., 1969, Exp. Cell Res., 5S:45S and Oftebro, R. and Nordbyc, K., 1 969, Exp. Cell Res., 58:459) were cultivated in medium Eagles minimum essential medium (MEM) supplemented with 10% fetal bovine serum and antibiotics. Cells were routinely grown as monolayers in tissue culture flasks. The cells were kept in continuous exponential growth by frequent rcculluring every second or third day. During reculturing, as well as during experiments, the cells were kept in humidified CCb incubators at 37°C.

In the experiments represented in this example, the protcinaceous CANP inhibitor was additionally purified by gel filtration chromatography. The molecular weight and purity were assessed by polyacrylamide gel electrophoresis, the molecular weight being on the order of 57.500 Dallon..

Cell survival studies were performed by adding inhibitors to exponentially growing, asynchronous cells 2 hours after seeding the cells into plastic Petri dishes at known cell densities. The inhibitor treatment period was 24 hours after which the dishes were" rinsed with warm (37°C) Hanks' balanced salt solution and fresh medium without inhibitor, ' was added . After 1 2 to 14 days of incubation (with a medium shift on day 7) colonies Qf cells were fixed in ethanol and stained with methylene blue. Only colonies containing more than 40 cells were scored as survivors. The percent cell survival is calculated relative to untreated, control dishes.

The results represented in Figure 1 show that calpain inhibitor 1 induced the greatest cytotoxic effect in cultures of human NH IK 3025 cervix carcinoma cells. Lcupeptin and E-64 induced little cytotoxic effect. I t appears that calpain inhibitor II induced slightly greater cytotoxicity than aprotinin.

However, far greater cytotoxicity based upon molar concentrations was induced by chromatographically purified endogenous inhibitor of CANPs, as shown in Figure 2.

Example 12 The effect of calpain inhibitor I and calpain inhibitor II on growth of Walker rat tumors // vivo.

Male VVistar rats were injected with 1 0 x 1 06 Walker tumor cells per rat at the inguinal region. Treatment was started when measurable tumors appeared. Each treatment group consist ed of 5 rats. Cal pain inhibitor I and calpain inhibitor II were formulated into egg lecitin and phosphatyl choli ne-containing li posomes. Rats were treated with 2 ml liposomal solution of inhibitors at a concentration of 1 3.5 mg/ml, equivalent to 27 mg/ l OOg rat, daily by intraperitoneal injection. Rats in the control group received daily injections of saline-filled liposomes. Tumor volume was determined by measuring two diameters with calipers. The data is presented in Figure 3 in which each data point represents the mean ± S .E. tumor volume measurements for 5 rat s.

The results show that 5 doses of calpain inhibitor I induced approximately 92% tumor regression. Calpain inhibitor II appears to induce an anticancer effect from the fourth dose.

Histological examination of the testis of the treated groups showed spennatogenctic arrest while the control group showed normal spermatogenesis. All other organs, including the liver, showed no cytotoxicity. e e ect o protease n ors on ce carcinoma cells // vitro. 1 2 3 Concentration of protease inhibitor (mM) Figure 1 The cflect of CANP inhibitor on cell survival of human NHIK 3025 cervix carcino cells //; vitro. The CANP inhibitor was chromatographically purified. 0.002 0.004 0.006 0.008 Concentration of protease inhibitor (mM) Figure 2 The effect of calpain inhibitors I and Π on growth of Walker rat tumors 1 2 3 4 Days after start of treatment Figure 3 102079/1

Claims (10)

1. The use of inhibitors of calcium activated neutral proteinases (CANPs) for the manufacture of pharmaceutical preparations for the treatment of tumours, especially cancer, viral diseases, AIDS and for the manufacture of anticonceptiva by blending the inhibitors with pharmaceutically acceptable solutions.

2. The use according to claim 1 , wherein the purified or unpurified inhibitor is a tetrameric protein having a molecular weight of approximately 240,000 based on elution from Sephadex G-200, is heat stable at neutral pH, is destroyed on digestion with trypsin, and is dissociated into subunits of a molecular weight of approximately 60,000 or lower by 0. 1 -lmM Ca2+, based on SDS-polyacrylamide gel electrophoresis, derivatives, analogues, active subunits, chemical or proteolytic fragments or related compounds thereof

3. The use according to claim 1, wherein the active compound is calpastatin having a molecular weight of approximately 70,000 Dalton being from, for example, erythrocytes.

4. The use according to claim 1 , wherein the active compound is calpastatin having a molecular weight of approximately 1 10,000 Dalton being from, for example, liver.

5. The use according to claim 2, wherein the inhibitor is an endogenous native inhibitor isolated from a biological source selected from the group consisting of erythrocytes, brain, cardiac muscle, lung, spleen, liver, skeletal muscle, kidney and testis.

6. The use according to claim 2, wherein the inhibitor is isolated from rabbit skeletal muscle.

7. The use according to claim 2, wherein the inhibitor is produced synthetically, or by bio- or genetechnological methods.

8. The use according to claim 1 , wherein the inhibitor is a tripeptide having the chemical composition N-acetyl-leu-leu-norleucinal.

9. The use according to claim 1 , wherein the inhibitor is a tripeptide having the chemical composition N-acetyl-leu-leu-methioninal.

10. A composition according to claim 1 in the form of a solution, powder, injection, tablet, capsule, pellet or a fast or sustained release preparation. 1 1. A method according to claims 1 wherein the CANP inhibitors are antimeiotic agents, against diseases caused by meiotic cell division. AGENT FOR APPL ICANT